METHODS FOR SCREENING ANTIESTROGEN COMPOUNDS

Abstract: The present invention provides novel assay methods for identifying compounds that may have both estrogen agonist and antagonist properties. In particular, the assay use cells comprising promoters having an AP1 site linked to a reporter gene. Compounds capable of inducing or blocking expression of the reporter gene can thus be identified. The compounds may be futher tested for the ability to modulate the standard estrogen response, as well.

Description: METHODS FOR SCREENING ANTIESTROGEN COMPOUNDS BACKGROUND OF THE INVENTION Many breast tumors require estrogens for tumor growth. Thus, treatment with antiestrogen compounds can slow or prevent tumor spread. Many antiestrogens however, show both estrogen antagonistic and agonistic activity. The nonsteroidal antiestrogen tamoxifen, for example, which is established as the treatment of choice for the endocrine therapy of advanced breast cancer, shows both agonistic and antagonistic activity. Sutherland, S. & Jackson, M. Cancer Treat . Revs . 15: 183-194 (1987). The agonistic activity of tamoxifen and other antiestrogens may have profound effects upon patients. For example, agonistic activity may have beneficial effects, such as preventing osteoporosis and reducing serum cholesterol. Love, et al . New Eng. J. Med. 326: 852-856 (1992). Love, et al . J. Natl . Cancer Inst. 82: 1327-1332 (1990). Conversely, agonistic activity may also be harmful. Tamoxifen for example sometimes increases endometrial tumor incidence lino, et al . Cancer Treat . & Res . 53: 228-237 (1991) or switches from inhibition to stimulation of estrogen dependent growth in breast tumor progression. Parker, M.G. (ed) Cancer Surveys 14: Growth Regulation by Nuclear Hormone Receptors. Cold Spring Harbor Laboratory Press (1992) . It is desirable to identify pure antiestrogens as they are anticipated to provide more rapid, complete or longer-lasting tumor responses. akeling, A. E. Breast Cancer Res . & Treat . 25: 1-9 (1993). For example, ICI 164,384, thought to be a pure antiestrogen, blocked MCF-7 cell invasion activity of a re-constituted basement membrane while estradiol and 4'-hydroxytamoxifen stimulated this activity suggesting that early treatment of breast cancer with a pure antiestrogen might be particularly beneficial in limiting tumor spread. Braacke, et al . , Br. J. Cancer 63: 867-872 (1991). Conversely, while pure antiestrogens appear preferable for cancer treatments, mixed agonist-antagonist compounds may be preferable for preventative treatment. Such compounds should combine sufficient antagonist activity on estrogen stimulated breast tumor growth while maintaining simultaneous agonist activity on bone density and serum lipid levels. Currently, antiestrogen compounds are screened with animal models such as the rat uterine test. These tests are cumbersome, slow, expensive and of uncertain application to humans because of differences between the human and rodent estrogen receptors. Alternatively, investigators have used human breast cancer cell lines and assayed their growth as stimulated by estrogens or alternatively used the response of native or transfected reporter genes as assays of estrogen response. These cell lines utilize reporter genes that indicate activity of antiestrogen compounds that is mediated through the classical estrogen response element. The prior art fails to provide methods for quickly and easily testing potential antiestrogen compounds for agnostic as well as antagonistic properties mediated through pathways other than the classical estrogen response pathway, that may affect, adversely or beneficially, their use in various therapeutic applications. This invention addresses these and other problems in the art. SUMMARY OF THE INVENTION The present invention provides methods for screening test compounds for the ability to activate or inhibit transcription through an indirect estrogen response or classical estrogen response. The indirect estrogen response is mediated by promoters comprising an API site and the classical estrogen response is mediated by promoters comprising a classical estrogen response element. Preferred API sites can be isolated from etalloprotease genes. Preferred classical estrogen response elements can be isolated from the Xenopus vitellogenin A2 gene. The methods typically use cells comprising an estrogen receptor and a promoter comprising an API site which regulates expression of a reporter gene. The cells are then contacted with the test compound and the expression of the reporter gene is detected. Cells used in the assay can be any cell which naturally expresses estrogen receptors or as the result of a transgene encoding the receptor. Preferred cells include MCF- 7, ERC1, ERC2 or ERC3. The reporter genes used to detect an estrogen response include genes encoding beta-galactosidase and bacterial chloramphenicol acetyl transferase. The promoters used may be those which naturally comprise API or estrogen receptor elements or the promoters may be genetically engineered to comprise those elements. The assays may be used with cells comprising promoters with an API site. Or alternatively a second set of cells with a promoter comprising a classical estrogen response element which regulates expression of a second reporter gene can be used. Alternatively, the two promoters and reporter genes can be in the same cells. In these assays the ability of test compound to induce or inhibit both the indirect and classical pathways can be determined.. When the methods are used to identify estrogen antagonists, the test compounds are contacted with the cells and a compound known to mediate an indirect estrogen response. The ability to inhibit the response is determined by detecting the expression of the reporter gene. Compounds known to mediate an indirect estrogen response include tamoxifen and estrogen at half maximal concentrations. The compounds can also be tested for the ability to induce or block the classical estrogen pathway, as well. The invention further provides compounds identified by the claimed methods. Also claimed are cells which over- express an estrogen receptor and comprise a promoter comprising an API site which regulates expression of a reporter gene. Exemplary cells include ERC1. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows estrogen stimulation mediated by an API site. (A) A consensus API site has ERE-like activity. Reporter genes, Δcoll73 and Δcoll60, were transfected into ERC1 cells (Kushner, et al . , Mol . Endocr±nol . , 4: 1465-1473 (1990) Δcoll73 consists of human collagenase promoter (-73 to +63) cloned upstream of the CAT gene. Angel, et al . Mol . Cell . Biol . , 7: 2256-2266 (1987). The position of the consensus API site is indicated. ΔcollδO lacked the API site (-73 to -60) . EREΔcoll60 contains an oligonucleotide corresponding to the frog vitellogenin A2 consensus ERE (Parker, M.G. (Ed) , Cancer surveys 14 Growth Regulation by Nuclear Hormone Receptors. Cold Spring Harbor Laboratory Press (1992)) immediately upstream of the collagenase sequences in Δcoll60. Typical results of CAT assays, normalized for transfection efficiency, following a single transfection are shown opposite. Each point is the mean value of triplicate assays, with standard errors. CAT activities from cells maintained in the absence of hormone are shown as white bars, those in the presence of a saturating concentration (100 nM) of estradiol as black bars. (B) Estrogen induction at the API site requires API proteins. Results of CAT assays prepared from F9 cells transfected with Δcoll73. Figure 2 shows activity of API sites in MCF-7 cells. Estrogen induction of API dependent reporter genes Δcoll73 and Δcoll60 was described above. APlΔTK-CAT consists of the collagenase API site cloned upstream of a herpes simplex virus TK promoter (-32 to +45) . TK-TATA lacks any known upstream transcription factor binding sites. CAT activities in the presence (black) and absence (white) of estrogen are shown opposite each reporter. Figure 3 shows agonism by antiestrogens at the API site. (A) CAT assays of ERC1 cells transfected with Δcoll73, Δcoll60 or EREΔcoll60 and incubated with saturating concentrations (lOOnM) of estrogen (black) , tamoxifen (shaded) and ICI (striped) or no hormone (white) . A single experiment is shown, where the activities are the mean of three separate points. (B) Dose dependence of antihormone agonism. ERC1, transfected with Δcoll73 were plated and exposed to a range of hormone concentrations. Each point is the mean of triplicate assays of CAT reactivity. (C) Antiestrogen activity in estrogen responsive cell lines. Results of representative CAT assays following transfection of Δcoll73 and ΔcollδO into MCF-7 cells and Δcoll73, Δcoll60 and EREΔcollδO into Ishikawa cells are shown. A single experiment is shown, where the activities are the mean of three separate points. Figure 4 shows antiestrogen action in transient transfections. (A) Δcoll73 was introduced into CHO cells, with varying quantities of ER expression vector HEO (Holinka, C.F. et al . , J. Steroid Biochem . , 25: 781-786 (1986) which is herein incorporated by reference) , normalized to lOμg with the parent expression vector, SG5. CAT activities were determined in the presence of hormones, as in Figure 3A. (B) Requirement of ER domains for hormone action. The structure of four derivatives of the human ER cDNA is shown schematically. The DNA binding domain is indicated with the striped box, the ligand binding domain is marked E2. Transactivation functions TAF-1 and TAF-2 are marked. The right panel shows the effect of hormone upon Δcoll73 in the presence of each mutant. Transfections were carried out as described by Kushner, et al . , 4: 1465-1473 (1990) which is herein incorporated by reference, in CHO cells, including 300ng of each ER expression vector. DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention provides an efficient way to screen large numbers of test compounds for those which have desirable properties for either the treatment or the prevention of various cancers (e.g. breast cancer, ovarian cancer, endometrial cancer) and other diseases (e.g. endometriosis) mediated by estrogen. The invention allows for screening of test compounds for agonistic as well as antagonistic activity. In addition, this invention provides methods of screening for novel types of antiestrogen compounds that block only the indirect estrogen response and do not block estrogen action at classical estrogen response elements. In animals and in man the balance between stimulatory and inhibitory activities of antiestrogens such as tamoxifen varies widely depending on the organ, cell or 80 specific protein measured as an indicator of estrogenic activity. This variety of effects is difficult to reconcile with the model of antagonism of estrogen receptor (ER) activity at classical estrogen receptor elements (EREs) as described in Beato, M. Cell , 56: 335-344 (1989) and Klein- 85 Hitpass, et al.. Nucleic Acids Res . , 16: 647-663 (1988) which are incorporated herein by reference. The present invention relies, in part, on the discovery that ERs may activate transcription by interaction with another response element, the API binding site, instead 90 of binding to EREs. This API mediated pathway, referred to here as the indirect estrogen response, may account for much of the agonistic properties of tamoxifen and other putative antiestrogens. A general description of the API site is found in Angel & Kann, Biochem . Biophyε . Acta . , 1072: 129-157 (1991) 95 and Angel, et al . , Cell , 49: 729-739 (1987), which are incorporated herein by reference. In the methods of the invention, both the classical estrogen response elements and the indirect estrogen response may be used to provide a screening system that detects both 100 estrogen antagonistic and agonistic activity. The methods typically comprise cultured cells that produce high levels of the human estrogen receptor. Preferred cells include MCF-7 cells or ERC1 cells described in Kushner et al . , Mol . Endocrinol . , 4:1465-1473 (1990), which is incorporated herein 105 by reference. ERC2 and ERC3 cells as described by Webb, et al . Mol . Endocrinol . , 6:157-167 (1993) which is herein incorporated by refererence. Other appropriate cells, including for example Ishikawa cells, and various breast cancer cells, which are 110 known to one of skill in the art. The invention is not limited to practice in mammalian cells and may be practiced, for example in yeast and insect cells. The cells may be modified to provide truncated or chimeric estrogen receptors as described in Berry, et al . , 115 E.M.B .O. J. , 9: 2811-2818 (1990) which is herein incorporated by reference. These modifications may result in increased estrogen affinity and increased sensitivity of the assay. In addition, these cells are transfected with reporter genes in which a response element (either the API 120 site or ERE) regulates expression of a reporter gene. Typically, two different reporter genes are used. One gene reports transcription induced by the classical estrogen response system, while the other gene reports transcription induced by the indirect estrogen response. The two reporter 125 genes and response elements are typically placed in separate cells, but the methods can also be used with both constructs in the same cell. The reporter gene for the classical estrogen response system contains an estrogen response element (ERE) 130 upstream of the target promoter and capable of regulating that promoter. In a preferred embodiment the ERE may be the consensus estrogen response element AGGTCACAGTGACCT from the Xenopus vitellogenin A2 gene. The particular ERE used in the cells is not a 135 critical aspect of the invention and the present invention is not limited to the use of this ERE.. Other EREs known to one of skill in the art can also be used. For instance, other sources of naturally occuring EREs include the B2 gene, the chicken ovalbumin gene, and the PS2 gene. Alternatively, non- 140 naturally occuring EREs may be inserted into particular promoters. The consensus ERE from the Xenopus vitellogenin A2 gene is widely used for this purpose, but other EREs may be used as well. The reporter gene for the indirect estrogen response 145 pathway contains an API site upstream of the target promoter and capable of regulating that promoter. The API site is a sites that are bound by API (the Jun and Fos proteins) or other members of that protein family. In a preferred embodiment, the consensus API site is TGA(C/G)TCA. 150 One of skill would recognize that the particular API site used is not a critical aspect of the invention. Any sequence capable of being bound by API or members of that family and regulating a promoter is suitable. This would include promoters which encompass a naturally occuring API 155 site. Typical promoters include, but are not restricted to metalloprotease genes such as stro elysin, gelatinase, matrilysin, and the human collagenase gene. Alternatively promoters may be constructed which contain a non-naturally occuring API or related binding site. 160 This facilitates the creation of reporter gene systems that are not typically found under the control of API. In addition, promoters may be constructed which contain multiple copies of the API site thereby increasing the sensitivity or possibly modulating the response the reporter gene system. 165 The present invention is not limited to a particular reporter gene. Any gene that expresses an easily assayable product will provide a suitable indicator for the present assay. Suitable reporter genes are well known to those of skill in the art. They include, for example, bacterial 170 chloramphenicol acetyl transferase (CAT) , beta-galactosidase, or luciferase. To screen a number of compounds for antiestrogen action, cells with high level expression of human estrogen receptors and harboring either response element and reporter 175 genes are exposed to doses of estrogen which give half maximal induction or less. In each case this will result in induction of several to hundreds of fold depending on the levels of estrogen receptor and the particular details of the reporter construction. This will be reflected in increases of the 180 reporter gene product, such as the CAT gene product which may be quantitated by enzymatic assay. The cells can be exposed to estrogens either growing in separate wells of a multi-well culture dish or for colorometic assay in a semi-solid nutrient matrix. The compounds to be tested are added to the culture 185 dish wells or to small wells made in the semi-solid matrix and the effect on the estrogen induction is assayed. An antiestrogen compound will reduce or abolish the estrogen induced increase in reporter gene activity. A hypothetical pure antiestrogen will block estrogen action with both types 190 of reporter genes and will have no ability to induce the reporter genes in the absence of estrogen. A mixed estrogen antagonist-agonist, will show some ability to induce the reporter genes, especially the reporter genes linked to API site. 195 In another embodiment, compounds which block the indirect pathway can be used to supplement tamoxifen or other antiestrogens in the treatment or prevention of breast cancer and other diseases mediated by estrogen. These compounds function to eliminate estrogenic agonistic activity of 200 antiestrogens. Second they may have uses by themselves. In particular, it may be advantageous to block some estrogen mediated pathological effects at indirect estrogen response elements while leaving the direct pathway active. Compounds that block the indirect pathway are useful as components of 205 combined oral contraceptives (COC) containing estrogens and progestins. A triple COC, containing estrogens, progestins, and a blocking compound would allow estrogen, either in the formulation or endogenous to act at the classical response elements, but would block action at the indirect response 210 elements. Thus, a triple COC functions as current COCs to prevent pregnancy, but may also provide protective effects against breast cancer. Typically, the reporter gene linked to the API site is activated, not with estrogen, but with an excess (10 times 215 the Kd) of tamoxifen, 4-hydroxy tamoxifen, or other antiestrogen. A library of compounds is searched for candidates that block or reduce reporter gene activation by the antiestrogen. The candidates are then tested with cells containing the reporter gene for the classical pathway to 220 confirm that they do not interfere with estrogen activation at an ERE. Compounds identified in the assays of the invention can be used in standard pharmaceutical compositions for the treatment of cancer, as components of oral contraceptives, or 225 any other application in which the modulation of estrogen activity is desired. The pharmaceutical compositions can be prepared and administered using methods well known in the art. The pharmaceutical compositions are generally intended for parenteral, topical, oral or local administration for 230 prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, and capsules. 235 Suitable pharmaceutical formulations for use in the present invention are found in Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985), which is incorporated herein by reference. A variety of pharmaceutical compositions comprising compounds of the 240 present invention and pharmaceutically effective carriers can be prepared. The following example is offered by way of illustration, not by way of limitation. EXAMPLE 245 The human collagenase gene, like other matrix metalloproteases, responds to API. Angel, et al . , Mol . Cell . Biol . , 7: 2256-2266 (1987). To test whether an API site could confer estrogen response the collagenase promoter was fused to the bacterial CAT gene (Δcoll73) and transfected into Chinese 250 Hamster Ovary cells that overexpress ER (ERC1) Kushner, et al . Mol. Endocrinol. 4: 1465-1473 (1990). Coll73 and Coll60, previously described by Angel, P. et al . , Mol . Cell . Biol . , 7 : 2256-2266 (1987) which is herein incorporated by reference, were modified by digestion with 255 Eco0109 and Ndel to remove an API site in the backbone of pUC and were consequently designated Δ. EREΔcollβO was prepared by ligation of a consensus ERE, AGGTCACAGTGACCT, into the Hindlll site upstream of the ΔcollδO promoter. ERC transfections and electroporations were carried out as 260 described in Kushner, et al . , Mol . Endocrinol . , 4: 1465-1473 (1990) which is herein incorporated by reference. F9 cell transfections were by calcium phosphate coprecipitation. Cells were seeded at 30% confluence upon 1.5 cmn dishes and transfected overnight with 5 mg reporter gene, 1 mg actin 265 B-HCG. Included in some transfections, as indicated, were lOOng of API expression vectors and lμg of ER expression vector HEO. Kumar, et al . , Cell , 51: 941-951 (1987) which is herein incorporated by reference. The human cfos expression 270 vector was BK28. Sassone-Corsi, et al . , Cell , 54: 553-560 (1988) which is herein incorporated by reference. The human c-jun expression vector was RSVc-Jun. Turner & Tjian, Science, 243: 1689-1694 (1989) which is herein incorporated by reference. The cells were glycerol shocked,re-fed and hormone 275 treated. All CAT assays were normalised to production of HCG as described in Kushner, et al . , Mol . Endocrinol . 4: 1465-1473 (1990) . Data are expressed as counts per minute of tritiated acetyl CoA converted per unit of HCG (CAT units) . Estradiol stimulated Δcoll73 ten fold (Fig. 1A) , 280 whereas a similar reporter in which the API site had been removed (ΔcollδO) , gave reduced basal activity and no estrogen response. Substitution of a classical ERE (Klein-Hitpass, et al . , Nucl . Acids Res . 16: 647-663 (1988)) for the API site (EREΔcollβO) , restored estrogen response, but not the elevated 285 basal activity. In F9 cells, which lack endogenous API activity (Chiu, et al . Cell , 54: 541-551 (1988) which is herein incorporated by reference) Δcoll73 failed to respond to estrogen in the presence of transiently transfected ER (Fig. 290 IB) . Estrogen activation could be restored by cotransfecting expression vectors for API proteins c-Jun and c-fos. Sassone- Corsi, P. et al . , Cell , 54: 553-560 (1988). Turner & Tjian, Science 243: 1689-1694 (1989). In parallel, EREΔcollδO was estrogen responsive, even in the absence of API, and Δcoll60 295 and remained unaffected by estrogen (data not shown) . Estrogen induction of the collagenase promoter therefore required both the API site and API proteins. To confirm that ER activates transcription through API sites under physiological conditions, we repeated the 300 study in MCF-7 (Fig. 2) , a human breast tumor cell line in which activity of the collagenase gene family is estrogen responsive and related to tumor invasiveness. Thompson, et al . , Cancer Res . 48: 6764-6768 (1988) which is herein incorporated by reference. 305 AP1ΔTK-CAT consists of the collagenase API site cloned upstream of a herpes simplex virus TK promoter (-32 to +45) . TK-TATA lacks any known upstream transcription factor binding sites. APlΔTK-CAT was constructed by ligation of an oligonucleotide spanning collagenase sequences -73 to -52 into 310 the Hindlll site upstream of a TK-CAT derivative which had been modified to remove the PUC API site. API TK-TATA was constructed by ligation of the same oligonucleotide into the Hindlll site of a CAT vector which contained TK sequences -32 to +45 and already lacked the PUC19 API site Leitmann, D.C. et 315 al . , Mol . Cell Biol . , 12: 1352-1356 (1992) which is herein incorporated by reference. MCF-7 transfections used the same protocol as ERC cells described in Kushner, et al . , Mol . Endocrinol . , 4: 1465-1473 (1990). Estrogen stimulated Δcoll73 activity four fold, and 320 removal of the API site abrogated estrogen responsiveness. The collagenase API site also conferred estrogen response upon the herpes simplex virus TK promoter and the isolated TK TATA box, neither of which was estrogen responsive alone. Thus, the API site was all that was required for estrogen response 325 on heterologous promoters. This type of estrogen induction may represent a far more common pathway than hitherto suspected. This pathway is referred to as the indirect estrogen response. The effects of antiestrogens on the indirect 330 estrogen response were examined next. All cells were transfected as described in Kushner, et al . Mol . Endocrinol . , 4: 1465-1473 (1990). Ishikawa cells, described by Holinka, et al . , J. Steroid Biochem . , 25: 781-786 (1986) which is incorporated by reference, were induced with lOnM PMA and lOμM 335 forskolin prior to estrogen and antiestrogen induction. Tamoxifen inhibits estrogen action at classical EREs by interfering with the estrogen dependent transcription activation function in the ER ligand binding domain. Berry, et al . , E.M.B .O. J. , 9: 2811-2818 (1990). It sometimes shows 340 partial agonism at classical EREs, which is attributed to activity of a cell type specific constitutive transactivation function in the amino terminus. Id. ICI inhibits a dimerizatioh function (Fawell, et al . Proc. Natl . Acad. Sci . U.S.A. 87: 6883-6887 (1990)), which prevents high affinity 345 interaction with EREs in target promoters, and does not show agonism in most assays. Wakeling, A. J. Steroid Biiochem. Molec. Biol . , 37: 771-775 (1990). Remarkably, both tamoxifen and ICI stimulated Δcoll73 expression five fold in ERCl cells (Fig.3A). Neither 350 affected Δcoll60. In contrast, EREΔcollβO showed estrogen, but not antiestrogens response indicating that antiestrogen agonism is specific to the API site. The dose response (Fig.3B) of each agonist was consistent with its affinity for the ER (Tora, et al . , E.M.B .O . J. , 8: 1981-1986 (1989) (InM 355 for estradiol, 10 nM for ICI and tamoxifen) . We also examined antiestrogen response in cells with endogenous ER. In MCF7, tamoxifen weakly stimulated Δcoll73 expression and ICI showed little effect (Fig.3C). A human endometrial cell line (Holinka, et al . , J. Steroid Biochem . , 25: 781-786 which is 360 herein incorporated by reference) , estrogen, tamoxifen and ICI were equally potent in eliciting an Apl site-specific two fold response but did not affect the classical ERE. The effect of transiently expressed ER mutants (Kumar, et al . Cell , 51: 941-951 (1987), Kumar & Chambon, 365 Cell , 55: 145-155 (1988) which is herein incorporated by reference) upon Δcoll73 in CHO cells was examined to determine which ER domains mediate an indirect response. Estrogen and tamoxifen activated the collagenase promoter (Fig.4A), although both were less effective with high amounts of 370 transiently transfected ER. ICI induction became more significant at high ER contents. This is consistent with observations that ICI reduces ER levels by reducing the half life of ER protein. Dauvois, et al . , Proc. Natl . Acad . Sci . U.S.A. , 89: 4037-44041 (1992). 375 The ER contains two transcriptional activation functions (Fig.4B), amino terminal constitutive (TAF-1) , and carboxyl terminal estrogen dependent (TAF-2) . Isolated TAF-1 (HE15) appears to give hormone independent activation of transcription and deletion of TAF-1 (HE19) reduced estrogen 380 response and abolished antiestrogen activity. Antiestrogen activity upon Δcoll73 in CHO cells therefore reflects TAF-1 activity. This agrees with suggestions that tamoxifen cannot activate TAF-2 (Berry, et al . (1990)) and further implies that ICI cannot activate this function. ICI agonism may also imply 385 that monomeric ER functions in this complex. Fawell, et al . (1990) . Deletion of the DNA binding domain (HE11) did not reduce hormone induction suggesting that the response is independent of DNA binding and rather mediated indirectly through API proteins. 390 In summary, indirect estrogen response is widely active, and antiestrogens are agonists of this pathway, it is possible that any of the well described agonist effects of tamoxifen reflects indirect estrogen response. Antiestrogens would have estrogenic activity on critical API regulated 395 target genes, hence growth and differentiated response, in cells in which ER and API proteins could interact. Changes in API during tumor progression could be particularly significant, and should be considered in models of antiestrogen resistance in breast cancer. Parker, et al . 400 Cancer Surveys , 14, Growth Regulation by Nuclear Hormone Receptors. Cold Spring Harbor Laboratory Press (1992) . Although the invention is described in some detail in the above description and examples for the purposes of clarity and understanding, it will be apparent that certain 405 changes and modifications can be made within the scope of the attached claims.

Claims: WHAT IS CLAIMED IS: 1. A method for screening a test compound for the ability to activate transcription through an indirect estrogen response, the method comprising: a) providing a cell comprising an estrogen receptor and a promoter comprising an API site which regulates expression of a reporter gene; b) contacting the cell with the test compound; and c) detecting the expression of the reporter gene. 2. A method of claim 1, wherein the cell is a MCF-7 cell. 3. A method of claim 1, wherein the cell over- expresses the estrogen receptor. 4. A method of claim 3, wherein the cell is an ERC1 cell. 5. A method of claim 1 wherein the reporter gene encodes beta-galactosidase. 6. A method of claim 1 wherein the reporter gene encodes bacterial chloramphenicol acetyl transferase. 7. The method of claim 1, wherein the promoter is genetically engineered to comprise an API site. 8. A method of claim 1, further comprising the steps of: a) providing a second cell comprising an estrogen receptor and a promoter comprising a standard estrogen response element which regulates expression of a second reporter gene; b) contacting the second cell with the test compound; and c) detecting the expression of the second reporter gene. 9. A method of claim 8, wherein the response element is from the Xenopus vitellogenin A2 gene. 10. A method of claim 1, wherein the cell further comprises a promoter comprising a standard estrogen response element which regulates expression of second reporter gene. 11. A method of claim 10 wherein the response element is from the Xenopus vitellogenin A2 gene. 12. An estrogen agonist identified by the method of claim 1. 13. A method for screening a test compound for the ability to inhibit transcription through an indirect estrogen response, the method comprising: a) providing a cell comprising an estrogen receptor and a promoter comprising an API site which regulates expression of a reporter gene; b) contacting the cell with the test compound and a compound known to mediate an indirect estrogen response; c) detecting the expression of the reporter gene. 14. The method of claim 13, wherein the compound known to mediate an indirect estrogen response is tamoxifen. 15. The method of claim 13, wherein the compound known to mediate an indirect estrogen response is estrogen at half maximal concentration. 16. A method of claim 13, wherein the cell is a MCF-7 cell. 17. A method of claim 13, wherein the cell over- expresses the estrogen receptor. 18. A method of claim 17, wherein the cell is an ERC1 cell. 19. The method of claim 13, wherein the promoter is genetically engineered to comprise an API site. 20. A method of claim 13, further comprising the steps of: a) providing a second cell comprising an estrogen receptor and a promoter comprising a standard estrogen response element which regulates expression of a second reporter gene; b) contacting the second cell with the test compound; and c) detecting the expression of the second reporter gene. 21. The method of claim 20, further comprising the step of contacting the second cell with a compound known to mediate a standard estrogen response. 22. A compound identified by the method of claim 13. 23. A composition comprising a cell which over- expresses an estrogen receptor and comprises a promoter comprising an API site which regulates expression of a reporter gene. 24. The composition of claim 23, 23 wherein the cell is ERC1.

Inventors: KUSHNER PETER, WEBB PAUL, KUSHNER, PETER, WEBB, PAUL
Applicants: UNIV CALIFORNIA, THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Application year: 1994
Main classification: C12Q1
IPC codes: A61K31, C07K14, C12N5, C12N15, C12Q1, G01N33